We are studying the Escherichia coli bacteriophage T4 as a model system for duplex DNA replication. Efficient DNA synthesis in vitro can be achieved with a mixture of seven purified proteins encoded by the phage. For strand displacement DNA synthesis on duplex templates, T4 DNA polymerase must be assisted by the gene 45 and 44/62 polymerase accessory proteins that increase the binding of the polymerase to the primer-template, and by the gene 32 protein that by itself destabilizes but does not melt native DNA. Our studies with the temperature-sensitive polymerase mutant ts CB120 demonstrate that a point mutation in the polymerase gene decreases the ability of the T4 replication complex to invade duplex templates. This defect in strand displacement may explain the decreased base substitution mutagenesis and increased frameshift mutagenesis that have been reported with the polymerase mutant in vivo. Discontinuous DNA synthesis on the lagging strand of the replication fork is initiated by pentaribonucleotide primers (pppACNNN) made by the T4 gene 41 and 61 proteins. Forty one Protein also has a helicase activity whose direction of unwinding supports the idea that it stimulates leading strand synthesis by destabilizing the helix ahead of the fork as it moves 5 feet to 3 feet toward the next primer site on the template for the lagging strand. In an effort to obtain the priming proteins needed to study coordination of leading and lagging strand synthesis, we have constructed plasmids containing T4 gene 41 that makes enzymatically active 41 protein in transformed cells. We are trying to identify and clone the T4 DNA region encoding gene 61. In collaboration with Dr. Gisela Mosig (Vanderbilt University), we are looking for site-specific initation of T4 DNA synthesis in extracts of T4-infected cells in which the modification of the host polymerase that inhibits initiation is blocked by mutations in T4 genes 33 and 55, and initiation from recombinational intermediates is blocked by mutations in T4 genes 46 and 47.